Replication of eukaryotic chromosomes is an ordered process involving the activation of multiple sets of replication origins during S phase. Within S phase, however, not all origins fire at the same time. It is this regulated firing of origins within S phase (the temporal program of origin activation) that has long fascinated us and that we propose to continue to study in budding yeast Saccharomyces cerevisiae. The four areas of proposed work include the following: (1) The S phase activators of Cdk1, Clb5p and Clb6p, are not equivalent with respect to origin firing. Clb6p, unlike Clb5p, is inefficient at activating late origins. We will test models for the mechanistic differences between Clb5p and Clb6p action with regard to late-firing origins. We will also explore the interaction between CDK and the other S phase kinase, Cdc7p. (2) What distinguishes early- from late-firing origins is unknown. We imagine there is an S phase clock that metes out critical initiation factors to different origins at different times during S phase. Reports in the literature suggest that cells treated with hydroxyurea (HU) or mutated in RAD53 show an altered clock with regard to late origins. We propose to explore the role of nucleotide pools and the S-phase checkpoint protein Rad53p in the S phase clock. (3) When an origin fires in S phase is due in large part to the chromosomal context of that origin. Origins near telomeres are late replicating; origins near centromeres are early replicating. We wish to test the idea that this temporal distinction between chromosome domains is important for chromosome maintenance and segregation. (4) The cis-regulatory elements responsible for origin timing only have been vaguely defined. We propose to extend our whole-genome replication studies to characterize late origins as a class in hopes of defining the cis-regulatory sequences and elucidating their mechanism(s) of action.